The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging
The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging
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DOI：
10.1529/biophysj.106.102459
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发表时间：
2007-10-01
影响因子：
3.4
通讯作者：
Gunzer, Matthias
中科院分区：
文献类型：
--
作者：
Niesner, Raluca;Andresen, Volker;Gunzer, Matthias
双光子显微镜对于深部组织和活体成像是不可或缺的。然而，目前基于单光束点扫描的技术已经达到了灵敏度和速度的极限，因为更高的性能需要更高的激光功率，导致样品降解。我们利用一个多焦点扫描头分裂成一行的64个焦点的激光束，允许样品照明在真实的时间在全激光功率。该技术需要电荷耦合器件场检测，而不是通过光电倍增管的常规检测。两种设置的光学性能的比较显示，在每个可测量的参数的功能等效性下降到200 mm的穿透深度，其中大多数实际的实验执行。光电倍增管检测的优势体现在成像深度>300 μ m，因为它们具有更好的信噪比，而只有电荷耦合器件才能实时检测快速过程（此处为血流）。我们还发现，这两种设备的点扩散功能强烈依赖于组织的构成和穿透深度。然而，采用深度校正的点扩散函数允许深层组织数据的三维去卷积达到类似于表面检测的图像质量。
Two-photon microscopy is indispensable for deep tissue and intravital imaging. However, current technology based on single-beam point scanning has reached sensitivity and speed limits because higher performance requires higher laser power leading to sample degradation. We utilize a multifocal scanhead splitting a laser beam into a line of 64 foci, allowing sample illumination in real time at full laser power. This technology requires charge-coupled device field detection in contrast to conventional detection by photomultipliers. A comparison of the optical performance of both setups shows functional equivalence in every measurable parameter down to penetration depths of 200 mm, where most actual experiments are executed. The advantage of photomultiplier detection materializes at imaging depths >300 mu m because of their better signal/noise ratio, whereas only charge-coupled devices allow real-time detection of rapid processes ( here bloodflow). We also find that the point-spread function of both devices strongly depends on tissue constitution and penetration depth. However, employment of a depth-corrected point-spread function allows three-dimensional deconvolution of deep-tissue data up to an image quality resembling surface detection.